Investigation on microfloral association in the roots of Macrotyloma uniflorum (Lam.) Verdc., a medicinally important tropical pulse-crop and their possible applications for crop improvement: a review

Macrotyloma uniflorum (Lam.) Verdc., an economically important medicinal plant belongs to the Leguminosae family. Being Afro-Asian origin, the plant has long tradition of uses. It is primarily used for its antiurolithiatic property although it has other medicinal uses. Being Leguminosae member, this plant can form rhizobial nodules and mycorrhizal associations. The rhizobia obtained from this plant are mostly belonged to Bradyrhizobium sp. Although, Rhizobium pusence has also been reported. Microbes as biofertilizers can be used to increase yield of this plant, as well as there is great potential for utilizing the microbes derived from this plant. In this review we aim to describe the plant M. uniflorum - its taxonomic characteristics, economic uses, putative active constituents, and beneficial microflora along with their applications

Endophytic microbes and their diverse beneficial aspects in various sectors: A critical insight

Endophytes are ubiquitous and grow in plant tissues without causing any harmful effects to the host. They include different groups of microorganisms such as bacteria, fungi and actinomycetes. Along with the host plants, the existing endophytes also co-evolve after a long relationship between them. Host plant-endophyte interaction is similar to that of plant growth promoting microbes as they induce the growth of the host plant and increase resilience against biotic and abiotic stresses. The interaction of plant endophytes at the molecular level and the effect of endophytes on host gene expression is a new field of study and are still rarely explored. Endophytes act as a promising resource of many invaluable bioactive secondary metabolites. Some of these bioactive compounds include alkaloids, polyphenols, sterols, xanthones, terpenoids, flavones, coumarins, polyketides, quinones, saponins, tannins, benzopyrones, dibenzofurans. These secondary metabolites are beneficial for agriculture, industrial and pharmacological purposes. As endophytes have beneficial effects in sustainable agriculture, plant disease management, pharmaceuticals, industry and environmental management in an eco-friendly way, thus improving the strategy of application of endophytes as biological agents in every aspect of our life is a very challenging field of research. Our aim in this present review is to focus on plant-endophyte interactions and their various dimensions in order to address some future possibilities for expediting the bioactive secondary metabolite production

In vivo analysis of trypanosome mitochondrial RNA function by artificial site-specific RNA endonuclease-mediated knockdown

Trypanosomes possess a unique mitochondrial genome called the kinetoplast DNA (kDNA). Many kDNA genes encode pre-mRNAs that must undergo guide RNA-directed editing. In addition, alternative mRNA editing gives rise to diverse mRNAs and several kDNA genes encode open reading frames of unknown function. To better understand the mechanism of RNA editing and the function of mitochondrial RNAs in trypanosomes, we have developed a reverse genetic approach using artificial site-specific RNA endonucleases (ASREs) to directly silence kDNA-encoded genes. The RNA-binding domain of an ASRE can be programmed to recognize unique 8-nucleotide sequences, allowing the design of ASREs to cleave any target RNA. Utilizing an ASRE containing a mitochondrial localization signal, we targeted the extensively edited mitochondrial mRNA for the subunit A6 of the F0F1 ATP synthase (A6) in the procyclic stage of Trypanosoma brucei. This developmental stage, found in the midgut of the insect vector, relies on mitochondrial oxidative phosphorylation for ATP production with A6 forming the critical proton half channel across the inner mitochondrial membrane. Expression of an A6-targeted ASRE in procyclic trypanosomes resulted in a 50% reduction in A6 mRNA levels after 24 h, a time-dependent decrease in mitochondrial membrane potential (ΔΨm), and growth arrest. Expression of the A6-ASRE, lacking the mitochondrial localization signal, showed no significant growth defect. The development of the A6-ASRE allowed the first in vivo functional analysis of an edited mitochondrial mRNA in T. brucei and provides a critical new tool to study mitochondrial RNA biology in trypanosomes

Engineering RNA endonucleases with customized sequence specificities

Specific cleavage of RNAs is critical for in vitro manipulation of RNA and for in vivo gene silencing. Here we engineer artificial site-specific RNA endonucleases (ASREs) to function analogously to DNA restriction enzymes. We combine a general RNA cleavage domain with a series of Pumilio/FBF (PUF) domains that specifically recognize different 8-nt RNA sequences. The resulting ASREs specifically recognize RNA substrates and efficiently cleave near their binding sites. ASREs can be devised to recognize and cleave various RNA target sequences, providing a useful tool to manipulate RNAs in vitro. In addition, we generate designer ASREs to specifically silence an endogenous gene in E. coli, as well as a mitochondrial-encoded gene in human cells, suggesting that ASREs can serve as a gene silencing tool with designed specificity

Treatment of Type 1 Myotonic Dystrophy by Engineering Site-specific RNA Endonucleases that Target (CUG) n Repeats

Myotonic dystrophy type 1 (DM1) is caused by the expansion of (CTG)n in the 3′ untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, which is transcribed as (CUG)n repeats that accumulate in the nucleus. The RNA repeats specifically sequester or change the expression levels of several RNA-binding proteins, leading to aberrant splicing of many target genes. In this study, we developed artificial site-specific RNA endonucleases (ASREs) that specifically bind and cleave (CUG)n repeats RNA. We have generated one ASRE that can target the expanded RNA repeats in DM1 patient cells and specifically degrade the pathogenic DMPK messenger RNAs with minimal effect on wild-type alleles. Such ASRE treatment significantly decreased the number of nuclear foci in DM1 patient cells and can reverse the missplicing of many genes affected in DM1 patients. Taken together, the application of ASRE provides a new route of gene therapy for DM1 treatment

The E3 Ubiquitin Ligase SCF(Cyclin F) Transmits AKT Signaling to the Cell-Cycle Machinery

The oncogenic AKT kinase is a key regulator of apoptosis, cell growth, and cell-cycle progression. Despite its important role in proliferation, it remains largely unknown how AKT is mechanistically linked to the cell cycle. We show here that cyclin F, a substrate receptor F-box protein for the SCF (Skp1/Cul1/F-box) family of E3 ubiquitin ligases, is a bona fide AKT substrate. Cyclin F expression oscillates throughout the cell cycle, a rare feature among the 69 human F-box proteins, and all of its known substrates are involved in proliferation. AKT phosphorylation of cyclin F enhances its stability and promotes assembly into productive E3 ligase complexes. Importantly, expression of mutant versions of cyclin F that cannot be phosphorylated by AKT impair cell-cycle entry. Our data suggest that cyclin F transmits mitogen signaling through AKT to the core cell-cycle machinery. This discovery has potential implications for proliferative control in malignancies where AKT is activated

VprBP/DCAF1 Regulates the Degradation and Nonproteolytic Activation of the Cell Cycle Transcription Factor FoxM1

The oncogenic transcription factor FoxM1 plays a vital role in cell cycle progression, is activated in numerous human malignancies, and is linked to chromosome instability. We characterize here a cullin 4-based E3 ubiquitin ligase and its substrate receptor, VprBP/DCAF1 (CRL4VprBP), which we show regulate FoxM1 ubiquitylation and degradation. Paradoxically, we also found that the substrate receptor VprBP is a potent FoxM1 activator. VprBP depletion reduces expression of FoxM1 target genes and impairs mitotic entry, whereas ectopic VprBP expression strongly activates a FoxM1 transcriptional reporter. VprBP binding to CRL4 is reduced during mitosis, and our data suggest that VprBP activation of FoxM1 is ligase independent. This implies a nonproteolytic activation mechanism that is reminiscent of, yet distinct from, the ubiquitin-dependent transactivation of the oncoprotein Myc by other E3s. Significantly, VprBP protein levels were upregulated in high-grade serous ovarian patient tumors, where the FoxM1 signature is amplified. These data suggest that FoxM1 abundance and activity are controlled by VprBP and highlight the functional repurposing of E3 ligase substrate receptors independent of the ubiquitin system

The splicing activator DAZAP1 integrates splicing control into MEK/Erk-regulated cell proliferation and migration

Alternative splicing of pre-mRNA is a critical stage of gene regulation in response to environmental stimuli. Here we show that DAZAP1, an RNA binding protein involved in mammalian development and spermatogenesis, promotes inclusion of weak exons through specific recognition of diverse cis-elements. The C-terminal proline-rich domain of DAZAP1 interacts with and neutralizes general splicing inhibitors, and is sufficient to activate splicing when recruited to pre-mRNA. This domain is phosphorylated by the MEK/Erk pathway and this modification is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Using mRNA-seq we identify endogenous splicing events regulated by DAZAP1, many of which are involved in maintaining cell growth. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect. Taken together, these studies reveal a molecular mechanism that integrates splicing control into MEK/Erk regulated cell proliferation

APC/C and SCF cyclin F Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry

The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems